Evaluation of Genetic Diversity in Quill Mites of the Genus Syringophiloidus Kethley, 1970 (Prostigmata: Syringophilidae) with Six New-to-Science Species

Simple Summary Morphology and barcode data were used to estimate the diversity and genetic variability of fourteen putative species of the genus Syringophiloidus Kethley, 1970. In most cases, both sources of information were consistent. The only exception was S. amazilia Skoracki, 2017, which according to our results is most likely a population of S. stawarczyki Skoracki, 2004, and probably should be treated as its junior synonym. The further findings of our study are six new-to-science species described herein. We indicate that both the host phylogeny and distribution can drive the evolution of quill mites. Our results increase the knowledge of quill mite diversity and provide some premises to formulate and further test evolutionary, ecological, and epidemiological inquiries. Abstract Quill mites (Acariformes: Syringophilidae) are poorly explored bird parasites. Syringophiloidus Kethley, 1970, is the most specious and widespread genus in this family. It is believed to contain mono-, steno- and poly-xenous parasites and thus seems to be an exemplary for studies on biodiversity and host associations. In this work, we applied the DNA barcode marker (mitochondrial cytochrome c oxidase subunit I gene fragment, COI) to analyze the species composition and host specificity of representatives of fifteen Syringophiloidus populations parasitizing fifteen bird species. The neighbor joining analyses distinguished thirteen monophyletic lineages, almost completely corresponding to seven previously known species recognized based on morphological features, and six new-to-science species. The only exception is S. amazilia Skoracki, 2017, which is most likely conspecific with Syringophiloidus stawarczyki Skoracki, 2004. The intraspecific distances of all species were not higher than 0.9%, whilst the interspecific diversity ranged from 5.9% to 19.2% and 6.3–22.4%, inferred as the distances p and K2P, respectively. Although all putative species (except S. amazilia) are highly supported, the relationships between them have not been fully resolved and only faintly indicate that both host phylogeny and distributions influence the phylogenetic structure of quill mite taxa.


Introduction
Quill mites (Acariformes: Syringophilidae) are widespread permanent bird ectoparasites.To date, 417 species have been described [1,2], although their actual number is estimated to be several times higher, probably reaching up to 5000 species [3].Although the knowledge about syringophilid diversity and host associations has been growing recently [4][5][6], they remain one of the least understood bird parasites.This is due to their small body size, poorly accessible habitats (bird's feather quill), and low prevalence [7].Further difficulties are caused by weakly informative morphology and relatively few diagnostic characters [8,9].Moreover, the vast majority of species were described only on the basis of female features, and the consequence is that males, nymphs, and larvae are virtually unidentifiable.To overcome the limitations of morphology, molecular methods have recently come into use in mite taxonomic studies [9][10][11].DNA barcoding is an approach employing a short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence.It is commonly used as an effective marker in the process of species identification in many groups of animals (Hebert 2003(Hebert , 2004) ) [12,13], including quill mites [14].Although only a very small fraction of those parasites have been barcoded so far, this approach has proven reliable in such systematic inquiries as female dimorphism or phenotypic plasticity [8,9].It has also been successfully used for the estimation of host spectrum [15] and cryptic species detection [16].
Precise and unambiguous species diagnosis is crucial for any other research, including that on quill mites' parasitological and epidemiological importance.This is particularly important in the context of recent reports that mites are the host of unique phylogenetic lineages of bacteria of the genera Wolbachia and Spiroplasma.In addition, the presence of Bartonella and Brucella taxa has been detected in syringophilids, which makes them potentially important in the process of circulation of pathogens among birds [17].
The Syringophiloidus Kethley, 1970, is the most specious and widely distributed genus of quill mites with 48 known species widespread around the world.This taxon has been recorded from 80 avian host species, belonging to 29 families and five orders [1,2].Since the species of this genus are known to have various (mono-, steno-and poly-xenous) associations with hosts, they seem to be a representative material for research on diversity and host associations.
In this paper, we supplement the morphology with DNA barcode coverage to evaluate the species composition and host specificity of representatives of fifteen Syringophiloidus populations parasitizing fifteen selected bird species.

Animal Material and Morphological Analysis
Mite material used in the study (Table 1) was acquired from several sources: (i) the collection of feathers deposited in the Smithsonian Institution, National Museum of Natural History, Department of Vertebrate Zoology, Division of Birds, Washington, DC, USA (USNM) (September 2014), and bird specimens originally collected in Gabon (2009), Namibia (2009), and Peru (2009); (ii) the Biocenter Grindel and Zoological Museum (University of Hamburg), and bird specimens originally collected in Tanzania; (iii) mite samples collected in Mexico (field no.SVM 08-0506-1/4) (2008) and Brazil (2010); (iv) mites obtained from dead birds (due to probable collisions with the window glass) found at the AMU campus, Pozna ń, Poland (2009).micrometers (µm).Idiosomal setation follows that of [18] with modifications adapted for Prostigmata by [19].The nomenclature of leg chaetotaxy follows that proposed by [20].
The application of this chaetotaxy to Syringophilidae was recently provided by [21] with a few changes by [22].Latin and common names of the birds follow [23].
Material depositories and abbreviations: AMU-Adam Mickiewicz University, Pozna ń, Poland; USNM-Smithsonian Institution, National Museum of Natural History, Washington, DC, USA.The voucher slides and corresponding DNA samples are deposited in the collection of the AMU and USNM under the identification numbers indicated below.The sequences are deposited in GenBank under accession nos.specified in Table 1.

Molecular Data and Analysis
Total genomic DNA was extracted from single specimens using DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) as described by [24].The COI gene fragment was amplified via PCR with degenerate primers: Aseq01F (GGAACRATATAYTTTATTTT-TAGA) and Aseq03R (GGATCTCCWCCTCCWGATGGATT) [9].PCR amplifications were carried out in 10 µL reaction volumes containing 5 µL of Type-it Microsatellite Kit (Qiagen), 0.5 µM of each primer, and 4 µL of DNA template using a thermocycling profile of one cycle of 5 min at 95 • C followed by 35 steps of 30 s at 95 • C, 1 min at 50 • C, and 1 min at 72 • C, with a final step of 5 min at 72 • C.After amplification, PCR products were diluted two-fold with water, and 5 µL of the sample was analyzed via electrophoresis on 1.0% agarose gel.Samples containing visible bands were purified with thermosensitive Exonuclease I and FastAP Alkaline Phosphatase (Fermentas, Thermo Scientific, Waltham, MA, USA).The amplicons (585 bp) were sequenced in one direction using the Aseq01F primer.Sequencing was performed with BigDye Terminator v3.1 on ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA).Sequence chromatograms were checked for accuracy and edited using Geneious R11 (Biomatters Ltd., Auckland, New Zealand).
Phylogenetic associations between the studied taxa were estimated with the neighbor joining (NJ) method implemented in MEGA7 [25].Support for the recovered trees was evaluated with 1000 (NJ) non-parametric bootstrap replicates [26].Pairwise distances between nucleotide COI sequences were calculated using Kimura's two-parameter (K2P) and distance p models [27] for all codon positions with MEGA7.Stibarokris phoeniconaias Skoracki and OConnor, 2010, was chosen as an outgroup to root the tree.Tree visualizations were prepared using tree editing tools in MEGA7 and Figtree v. 1.4.2-[28](http://tree.bio.ed.ac.uk/)URL (accessed on 15 October 2023).

Molecular Analysis
We provided DNA barcode coverage for the representation of fifteen populations of Syringophiloidus ssp.recorded from fifteen bird species.The COI alignment was 552 bp long and comprised 43 sequences of Syringophiloidus mites (ingroup) and one sequence of Stibarokris phoeniconaias Skoracki & OConnor, 2010 (outgroup).The number of sequences obtained from each mite population varied from 1 to 10.The alignment contained 242 variable sites, 196 of which were parsimony informative.
The neighbor joining phylogenetic analyses (K2P and distance p) distinguished thirteen monophyletic lineages, among which seven lineages exactly correspond to seven previously known and species that are morphologically distinguished here.The only exception in the obtained pattern is presented by S. amazilia, which is very close to that of S. stawarczyki and most likely represents a population or subspecies of this species (Figures 1 and A1).
The neighbor joining phylogenetic analyses (K2P and distance p) distinguished thirteen monophyletic lineages, among which seven lineages exactly correspond to seven previously known and species that are morphologically distinguished here.The only exception in the obtained pattern is presented by S. amazilia, which is very close to that of S. stawarczyki and most likely represents a population or subspecies of this species (Figures 1 and A1).This assumption is also supported by the genetic distance between the two populations (1.4 and 1.5% of distance p and K2P) (Table 2), which is lower than that between bihost S. plocei populations (2.1%) and comparable to the previously reported intraspecific values within other quill mites [8].Although all putative species are highly supported with bootstrap values (100%) and, as they predictably delineate the morphospecies, the relationships between them have not been fully resolved and only weakly suggest various evolutionary scenarios.
The genetic distances were compared at intra-and inter-specific levels according to both the distance p and the K2P model.The integrity and separateness of particular taxa were proven for almost all populations resulting in the recognition of seven previously known species and six species new to science.The intraspecific distances of all species were not higher than 0.9%, whilst the interspecific diversity ranged from 5.9% to 19.2% and 6.3-22.4% for genetic distances p and K2P, respectively (Table 2).This assumption is also supported by the genetic distance between the two populations (1.4 and 1.5% of distance p and K2P) (Table 2), which is lower than that between bihost S. plocei populations (2.1%) and comparable to the previously reported intraspecific values within other quill mites [8].Although all putative species are highly supported with bootstrap values (100%) and, as they predictably delineate the morphospecies, the relationships between them have not been fully resolved and only weakly suggest various evolutionary scenarios.
The genetic distances were compared at intra-and inter-specific levels according to both the distance p and the K2P model.The integrity and separateness of particular taxa were proven for almost all populations resulting in the recognition of seven previously known species and six species new to science.The intraspecific distances of all species were not higher than 0.9%, whilst the interspecific diversity ranged from 5.9% to 19.2% and 6.3-22.4% for genetic distances p and K2P, respectively (Table 2).For females (holotype and three paratypes; range in parentheses) (Figure 2A-E), the total body length is 605 (600-620).For Gnathosoma, the infracapitulum is punctate.Each medial and lateral branch of peritremes has 2-3 and 9-11 chambers, respectively (Figure 2C).The stylophore is punctate and has a body length of 165 (150-155).For Idiosoma, the propodonotal shield is rounded anteriorly and sparsely punctate on the entire surface.The length ratio of setae vi:ve:si is 1:1:3.3-4.6.The hysteronotal shield is clearly visible and punctate in anterior and posterior parts.The pygidial shield is punctate and distinctly sclerotized in the area bearing bases of setae f1 and f2.Setae f1 and h1 are subequal in length.The length ratio of setae ag1:ag2:ag3 is 1.1:1:1.2.For Legs, Coxal fields I-IV are sparsely punctate.Setae 3c is 3.4-3.6times longer than 3b.Fan-like setae p' and p" of legs III-IV have seven tines (Figure 2E).Setae tc" is 1.For males (paratype) (Figure 3A-E), the total body length is 400.For Gnathosoma, the infracapitulum is apunctate.The stylophore is apunctate and 130 long.Each medial branch of peritremes has four chambers, and each lateral branch has 10 chambers (Figure 3C).For Idiosoma, the propodonotal shield is weakly sclerotized, bearing bases of setae vi, ve, si, se, and c1, and sparsely punctate near bases of setae vi, ve and si.Striation is clearly visible on the entire surface.The length ratio of setae ve:si is 1:1.The hysteronotal shield is weakly sclerotized, and the striae are visible, not fused to a pygidial shield, and apunctate.Setae d1, d2, and e2 are subequal in length.The pygidial is shield small, restricted to bases of setae f2 and h2, and to the genito-anal region or only to the genito-anal region; it is apunctate.Genital setae g1 is situated anterior to the level of setae g2, and both pairs are subequal in length.Pseudanal setae ps1 and ps2 are subequal in length.Length ratios of setae ag1:ag2 and f2:h2 are 1.3:1 and 1:11.5, respectively.Coxal fields I-IV are punctate.Setae 3c is four times longer than 3b.For legs, fan-like setae p' and p" of legs III and IV have 6 tines (Figure 3E).The length ratio of setae tc'III-IV:tc"III-IV is 1:1.7.The lengths of setae are as follows: ve 15, si 15, se 100, c1 100, c2 55, d1 13, d2 13, e2 13, f2 10, h2 115, ag1 45, ag2 35, 3b 10, 3c 40, l'RIII 13, l'RIV 15 tc'III-IV 15, and tc"III-IV 25.

Host and Distribution
Birds of the family Passerellidae: the white-headed brushfinch, Atlapetes albiceps (Taczanowski) from Peru.

Etymology
The name is taken from the generic name of the host and is a noun in apposition.

Etymology
The name is taken from the generic name of the host and is a noun in apposition.

Host and Distribution
Birds of the family Ploceidae: the white-browed sparrow-weaver, Plocepasser mahali Smith from Namibia.

Host and Distribution
Birds of the family Hirundinidae: the plain martin, Riparia paludicola (Vieiilot) from Namibia.

Etymology
The name is taken from the specific name of the host and is a noun in the genitive case.Syringophiloidus ripariae sp.n.

Host and Distribution
Birds of the family Hirundinidae: the sand martin Riparia riparia (L.) from Poland.

Type Material
The type material was a female holotype from the quill of the sand martin, Riparia riparia (L.) (Passeriformes: Hirundinidae), Pozna ń, POLAND, 52.4672007265976, 16.924954974622207, April 2009, coll.Glowska E.; the voucher and DNA code are as follows: EG079 (holotype).DNA barcode GenBank accession numbers are specified in Table 1.

Type Material Deposition
The holotype was accidentally crushed after species diagnosis was carried out and before the specimen was drawn.

Etymology
The name is taken from the specific name of the host and is a noun in the genitive case.

Host and Distribution
Birds of the family Trochilidae: the white-bellied emerald, Amazilia candida (Bourcier and Mulsant), from Mexico [34].

Remark
Our results revealed that S. stawarczyki and S. amazilia are conspecific, and as a consequence, S. amazilia could be treated as a junior synonym of S. stawarczyki.Although our results are precise, they are based on a relatively small sample.This is due to the limited availability of the mite material.For this reason, we do not formally synonymize these species, but only formulate a premise for further systematic research on the populations covering a more significant number of individuals.

Material Examined
Four females from the quill of the hooded crow Corvus corone cornix L. (Passeriformes: Corvidae) were used, and the material obtained from dead birds (due to probable collisions with a window) was found on the AMU campus, Pozna ń, Poland (8 May 2009), coll, Glowska E. Specimen vouchers and DNA codes are as follows: EG519 and EG522.DNA barcode GenBank accession nos.are given in Table 1.

Material Examined
Five females from the quill of the redwing Turdus iliacus L. (Passeriformes: Turdidae) made up the material examined, and the material was obtained from dead birds (due probable collision with glass) found on the AMU campus, Pozna ń, Poland (16 July 2009), Birds of the family Ploceidae: the grey-headed social weaver, Pseudonigrita arnaudi (Bonaparte), from Tanzania (Glowska et al., 2012) [10].

Material Examined
Four females from the quill of the frozen specimen of the grey-headed social weaver, Pseudonigrita arnaudi (Bonaparte) (Passeriformes: Ploceidae), were examined; the bird host was initially collected from the wild in Tanzania and imported to Hamburg in 1990 where it was housed in the Biozentrum Grindel and Hamburg Zoological Museum in the University of Hamburg, Germany, coll.E. Glowska, November 2010.
Specimen vouchers and DNA codes are as follows: EG545-547.DNA barcode GenBank accession nos.are given in Table 1.

Discussion
Both topologies of the phylogenetic trees and genetic distances revealed thirteen strongly supported monophyletic lineages which are in most cases in accordance with the morphological identifications.The only exception is S. amazilia, which very close to the S. stawarczyki clade and most likely represents a population of this species.This result is further supported by the genetic distance between the two lineages (1.4% and 1.5% of distances p and K2P, respectively), which is lower than that between bihost S. plocei populations (2.1%) and comparable to the previously reported intraspecific values within other quill mites [8,16].Also, a morphological analysis of the type material of both species showed that they are almost indistinguishable and share most diagnostic characteristics (both qualitative and quantitative).The differences between the alleged "species" are very subtle and manifest only in the length of setae d2 (135-170 in females of S. amazilia vs. 115-125 in S stawarczyki), f2 (175 vs. 220), ag1 (105-120 vs. 130-135), and ag2 (100-110 vs. 125-135).It is very likely that the differences are caused by the fact that both species were described based on a few specimens only (seven and three females of S. amazilia and S. stawarczyki, respectively) [29,34].This is a common practice when researchers work with hard-to-reach and low-prevalence material.It seems, however, that more individuals' availability would fill the metric data gap between S. amazilia and S. stawarczyki and show the continuity of the divergent characters.The presence of the same mite species on two phylogenetically distant hosts (representatives of different orders, i.e., Apodiformes and Passeriformes) can be explained by horizontal transfer since the ranges of both hosts overlap in Central America.At the moment, we do not have sufficient data to point the direction of the transfer.To carry this out, more individuals representing more populations of both hosts should be analyzed The cases of the host switching of quill mites have already been reported and our result supports the earlier assumption that this phenomenon is not incidental but rather one of the possible scenarios for the dispersion and evolution of this group of parasites [16,37].
In all other cases, the analysis of molecular data (NJ and genetic distances) confirmed the morphological separateness of previously known and newly described species.The intraspecific distances of all tested taxa were not higher than 0.9% and were comparable to the interpopulation values, i.e., 1.5% between S. plocei from the vieillot's black weaver and village weaver.All these values are similar to those previously observed in other stenoxenous quill mites (0.0-2.3) [8,14].Also, interspecific diversity, which ranged from 5.9% to 19.2% and 6.3-22.4% based on distance p and K2P, respectively (Table 2), is comparable to that among the species in other previously barcoded syringophilid genera [16].
Although all putative species (except S. amazilia) are highly supported with bootstrap values (100%), the relationships between them have not been fully resolved and only faintly indicate that both the host phylogeny and distributions may influence the phylogenetic structure of mites.For example, S. ripariae sp.n. from Poland and S. paludicolae sp.n. from Namibia were both recorded from hirundinid birds.Their populations show clear intraspecific integrity as well as species separateness measured via genetic distance (16.3% and 14.5% of K2P and p, respectively).Even though both species come from geographically distant locations, they form a sister group on the phylogenetic tree.This may suggest a parallel evolution of mites with avian hosts.Another example of a co-phylogenetic relationship is shown by S. plocei found on two ploceid species in Namibia.This clade forms a sister group with S. pseudonigritae, a parasite of another ploceid bird, the grey-headed social weaver in Tanzania.This result confirmed our earlier observations for these taxa (Glowska et al. 2016) [15].Another factor that may shape the phylogenetic structure of mites is geographical distribution.Two species, S. glandarii and S. parapresentalis, form a statistically well-supported sister group.Although they were obtained from birds from different families (Corvidae and Turdidae, respectively), they have a common location (Poland).Analogously, two species parasitize separate bird orders, S. calamonastes sp.n. and S. picidus form the "Namibian cluster".The same can be observed with the clearly distinct clade represented by mites from Mexico and South America (S.atlapetes; S. Stawarczyki-S.amazilia).
In this work, we used morphological and barcode data to estimate the diversity and genetic variability of fifteen populations of the genus Syringophiloidus.In most cases, both sources of information were consistent.The only exception was S. amazilia, which seems to be a population of S. stawarczyki and formally should be treated as its junior synonym.The further findings of our study are six now-to-science species, described herein.We indicate that both host phylogeny and distribution can drive the evolution of quill mites.However, we treat our results as a starting point for further in-depth research on these issues.Our results increase the knowledge about mite diversity and demonstrate the usefulness of the parallel use of morphological and molecular methods in solving systematic puzzles in this group of parasites.

Conclusions
Even though there has been progress in understanding quill mite systematics, little is known about their global diversity and host associations.This is mainly due to the weakly informative morphology and relatively few diagnostic characters.To address this challenge, a combination of classical morphology and DNA barcodes is used to increase the efficiency of species identification.This approach has been proven to be a reliable tool for this purpose, regardless of sex or developmental stage.It is also helpful for estimating genetic diversity and host specificity issues or revealing phenomena resulting from the incorrect interpretation of morphological characters, such as phenotypic plasticity, polymorphisms, or cryptic species.
Accurate species diagnosis is essential for further research, particularly in understanding quill mites' epidemiological importance.Recent reports suggest that mites host unique phylogenetic lineages of bacteria, such as Wolbachia and Spiroplasma.Additionally, they are believed to spread diseases by ingesting food (sucking the host's bodily fluids), although their epidemiological significance has not yet been well studied.Our findings contribute to knowledge about mite diversity and provide a basis for further evolutionary, ecological, and epidemiological investigations.

Figure 1 .
Figure 1.Neighbor joining phylogenetic tree of the Syringophiloidus species based on the K2P model.The tree was constructed in Mega v.7.and rooted by Stibarokris phoeniconaias.

Figure 1 .
Figure 1.Neighbor joining phylogenetic tree of the Syringophiloidus species based on the K2P model.The tree was constructed in Mega v.7.and rooted by Stibarokris phoeniconaias.

Figure A1 .
Figure A1.Neighbor-joining phylogenetic tree of the Syringophiloidus species based on the distance p model.The tree was constructed in Mega v.7.and rooted by Stibarokris phoeniconaias.

Table 1 .
Mites and sequences used in the molecular study.

Table 2 .
Estimates of evolutionary divergences between COI sequences of Syringophiloidus populations based on K2P (and p) distances.